CN114605493B - Synthesis method of antibody-coupled drug intermediate SET0526 - Google Patents
Synthesis method of antibody-coupled drug intermediate SET0526 Download PDFInfo
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- 229940079593 drug Drugs 0.000 title claims abstract description 10
- 239000003814 drug Substances 0.000 title claims abstract description 10
- 238000001308 synthesis method Methods 0.000 title abstract description 9
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims abstract description 48
- 150000001875 compounds Chemical class 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims abstract description 12
- 239000002253 acid Substances 0.000 claims abstract description 11
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims abstract description 6
- HOYRZHJJAHRMLL-UHFFFAOYSA-N 2,6-dinitro-p-cresol Chemical compound CC1=CC([N+]([O-])=O)=C(O)C([N+]([O-])=O)=C1 HOYRZHJJAHRMLL-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000007821 HATU Substances 0.000 claims abstract description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims abstract description 6
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- 239000002904 solvent Substances 0.000 claims description 32
- 239000012071 phase Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000004108 freeze drying Methods 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 7
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 238000010189 synthetic method Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- 229940049595 antibody-drug conjugate Drugs 0.000 description 9
- 238000002514 liquid chromatography mass spectrum Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 239000003053 toxin Substances 0.000 description 5
- 231100000765 toxin Toxicity 0.000 description 5
- 108700012359 toxins Proteins 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 4
- 238000010511 deprotection reaction Methods 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 238000007086 side reaction Methods 0.000 description 4
- 101710112752 Cytotoxin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000000611 antibody drug conjugate Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 231100000599 cytotoxic agent Toxicity 0.000 description 3
- 239000002619 cytotoxin Substances 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- 231100000782 microtubule inhibitor Toxicity 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000003915 DNA Topoisomerases Human genes 0.000 description 2
- 108090000323 DNA Topoisomerases Proteins 0.000 description 2
- 239000012623 DNA damaging agent Substances 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 2
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 102000007537 Type II DNA Topoisomerases Human genes 0.000 description 2
- 108010046308 Type II DNA Topoisomerases Proteins 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- KKTCWAXMXADOBB-UHFFFAOYSA-N azanium;hydrogen carbonate;hydrate Chemical compound [NH4+].O.OC([O-])=O KKTCWAXMXADOBB-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940125898 compound 5 Drugs 0.000 description 2
- 208000012839 conversion disease Diseases 0.000 description 2
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 2
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical compound NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 229930126263 Maytansine Natural products 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000012668 chain scission Methods 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019439 ethyl acetate Nutrition 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- CSRZQMIRAZTJOY-UHFFFAOYSA-N trimethylsilyl iodide Chemical compound C[Si](C)(C)I CSRZQMIRAZTJOY-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention provides a synthesis method of an antibody coupled drug intermediate SET0526. The synthesis method comprises the following steps: preparing MA-PEG4-VC-PAB by using compounds MA-PEG4-C 2H4 COOH, HATU, VC-PAB and DIPEA; MA-PEG4-VC-PAB-PNP is prepared from MA-PEG4-VC-PAB, DNPC and DIPEA; PNU-Acid is prepared from PNU-TBS, TBAF and sodium periodate; PNU-EDA-Fmoc was prepared with PNU-Acid, HOAt, prBrOP, EDA-Fmoc and DIPEA; PNU-EDA was prepared with PNU-EDA-Fmoc and DBU; SET0526 was prepared with MA-PEG4-VC-PAB-PNP, PNU-EDA, HOBt, DIPEA. The synthesis method of SET0526 has high yield and conversion rate, is easy to amplify and is convenient for commercial production.
Description
Technical Field
The invention relates to a method for synthesizing a compound, in particular to a method for synthesizing an antibody coupled drug intermediate SET0526, and belongs to the technical field of small molecule drug synthesis.
Background
The antibody drug conjugate (Antibody drug conjugate, ADC for short) is a novel anti-tumor drug, and the principle is that cytotoxin is connected to an antibody, and the antibody is used for recognizing a specific antigen on the surface of a cancer cell and entering the cancer cell through endocytosis, so that the cytotoxin is transported to a target point, and the purpose of targeted treatment of malignant tumor is achieved. Compared with the traditional small molecule antitumor drugs, the ADC has more specificity and effectiveness because of the targeting recognition property of the antibody and the high activity of the toxin.
The ADC comprises three distinct components, namely an antibody, a linker and a cytotoxin. The antibody realizes targeting, the linker ensures the stability of the ADC in the blood transport process, and the toxin plays a role in killing cancer cells after reaching an action target point. Toxins suitable for use in ADCs are classified into microtubule inhibitors (Microtubule inhibitors), DNA damaging agents (DNA DAMAGING AGENTS), RNA polymerase inhibitors (RNA polymerase inhibitors), and the like, depending on the mechanism of action.
Currently, the toxins used in ADCs on the market and in clinical trials are mainly microtubule inhibitors, mainly including compounds designed for DNA topoisomerase I (topoisomerase I) inhibitors, such as Dxd, and compounds designed based on maytansinoids (MAYTANSINE-based), such as DM1 and DM4; the DNA topoisomerase II (topoisomerase II) inhibitor designed compound, such as a highly potent anthracycline metabolite PNU-159582, has excellent cytotoxicity. In the aspect of the linker, non-cleavable type such as Valine-citrulline (Valine-CITRILINE) and cyclohexylformic acid (MCC) are mainly used, and the drug is still active after lysosomal hydrolysis and is combined with a certain amino acid residue through a connecting region.
There are various ways of forming antibody drug conjugates. The antibody can be chemically coupled with a drug linker through amino or sulfhydryl groups on the antibody, or modified, and then chemically coupled with the drug linker or enzymatically coupled after a specific functional group is introduced on the antibody.
Wherein, the antibody drug toxin molecule MA-PEG4-vc-PAB-EDA-PNU 159582 (SET 0526, structure is as follows) is a novel intermediate for antibody coupling drugs.
The synthetic method of SET0526 we developed includes the following three routes.
Route one:
in the development of the first route, the synthesis yield of the compound 5 is low, a lot of side reactions occur in the reaction, the total yield is low, and the large-scale production is difficult.
Route two:
Route two: since the synthesis yield of compound 5 is low and there are many side reactions occurring in the reaction in scheme one, we changed the synthesis strategy and then obtained compound 9 in high yield by deprotection of ethylenediamine protected with protecting groups.
In actual synthesis, the reaction conversion and yield of Boc protected 9-1 is high, but Boc protected 9-1 attempts different deprotection reagents such as: HCl/dioxane, HCl/EtOAc, HCl/Et2O, TFA, tosOH/THF-CH 2Cl2、Me3SiI/CHCl3 or CH 3 CN, all in low yields, and many side reactions (hydrolysis, chain scission, etc.) occur in low yields;
after the conversion of the protecting group to Fmoc, the reaction conversion and yield of Fmoc-protected 9-1 were also high, but Fmoc-protected 9-1 was tried with different deprotection reagents during the deprotection stage such as: the reaction yields of diethylamine, diisopropylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU), piperidine, ethanolamine, cyclohexylamine, morpholine, pyrrolidone 2 and the like are very low, and a plurality of side reactions occur in the reaction, and the yield is still very low.
So the final overall yield of the second route is still low and the scale-up production is difficult.
Disclosure of Invention
In order to solve the problems in the developed technology, we have developed a route three, namely a route method for the key protection of the invention; the invention aims to provide a synthesis method of high-efficiency SET0526 with high yield and high conversion rate.
In order to achieve the technical purpose, the invention provides a synthesis method of an antibody coupled drug intermediate SET0526, which comprises the following steps:
Stirring the compound MA-PEG4-C 2H4 COOH and HATU in a first solvent, cooling to 0-5 ℃ with ice water, adding the compound VC-PAB and DIPEA, reacting for 2-3 h under the protection of nitrogen, passing the reaction solution through a reverse phase column, and purifying to obtain the compound MA-PEG4-VC-PAB;
Dissolving the compound MA-PEG4-VC-PAB in a first solvent, adding DNPC and DIPEA, reacting for 14-16 hours at the room temperature of 20-25 ℃, purifying by medium-pressure reverse phase column chromatography, and obtaining the compound MA-PEG4-VC-PAB-PNP;
Dissolving a compound PNU-TBS in a second solvent, cooling to-5-0 ℃, adding TBAF, reacting for 20-30min, adding water and trifluoroethanol, adding sodium periodate, heating to 0-5 ℃ for reacting for 2-4h, detecting that the raw materials are completely reacted, directly concentrating and freeze-drying the reaction solution, and obtaining a crude compound PNU-Acid;
Dissolving the crude compound PNU-Acid in a third solvent, sequentially adding HOAt and PrBrOP, cooling by ice bath at 0-5 ℃, stirring for 5-0min, adding EDA-Fmoc and DIPEA, naturally heating to room temperature at 20-25 ℃ for reacting for 2-4h, performing aftertreatment, purifying by medium-pressure reverse phase column chromatography, and freeze-drying to obtain the compound PNU-EDA-Fmoc;
Adding the PNU-EDA-Fmoc compound into a fourth solvent for dissolution, cooling by ice bath at 0-5 ℃ and stirring for 5-0min, adding DBU, naturally heating to room temperature and stirring for 1-3h, carrying out aftertreatment, concentrating the reaction solution, purifying by medium-pressure reverse phase column and freeze-drying to obtain the PNU-EDA compound;
Dissolving the compounds MA-PEG4-VC-PAB-PNP and PNU-EDA in a third solvent, adding HOBt and DIPEA, naturally heating to room temperature of 20-25 ℃ for reacting for 2-4h, and performing post-treatment to obtain SET0526 MA-PEG4-VC-PAB-EDA-PNU 159582.
The synthesis method specifically comprises the following steps:
in one embodiment of the present invention, when preparing MA-PEG4-VC-PAB, the mixed molar ratio of MA-PEG4-C 2H4 COOH, HATU, first solvent, VC-PAB and DIPEA is 1.0eq:1.0eq-1.5eq:10V-20V:1.0eq:2.0eq-2.5eq.
In one embodiment of the present invention, when preparing MA-PEG4-VC-PAB-PNP, the mixed mole ratio of MA-PEG4-VC-PAB, DNPC and DIPEA in the first solvent is 1.0eq:10V-20V:1.0eq-1.5eq:2.0eq-2.5eq.
In a specific embodiment of the invention, the mixed molar ratio of PNU-TBS, in the second solvent, TBAF, water, trifluoroethanol, sodium periodate is 1.0eq:10V-20V:1.0eq-3.0eq:10V-20V:1.0eq-1.1eq.
In one embodiment of the present invention, PNU-EDA-Fmoc is prepared with a mixed molar ratio of PNU-Acid, third solvent, HOAt, prBrOP, EDA-Fmoc and DIPEA of 1.0eq:10V-20V:1.0eq-2.0eq:1.0eq-2.5eq:1.0eq-2.5eq:3.0eq-6.0eq.
In a specific embodiment of the invention, the mixed molar ratio of PNU-EDA-Fmoc, fourth solvent, DBU is 1.0eq:10V-30V:1.0eq-3.0eq.
In a specific embodiment of the invention, the mixed molar ratio of MA-PEG4-VC-PAB-PNP, PNU-EDA, third solvent, HOBt, DIPEA is 1.0eq:1.0eq-2.0eq:10V-30V:1.0eq-2.0eq:2.0eq-4.0eq.
In one embodiment of the present invention, when preparing SET0526, the post-treatment comprises the steps of:
High pressure liquid phase preparation, 234nm, flow rate 50mL/min,0.05% NH 4HCO3 water/acetonitrile, lyophilization.
In a specific embodiment of the invention, the first solvent is DCM, THF or DMF.
In a specific embodiment of the invention, the second solvent is DCM or THF.
In a specific embodiment of the present invention, the third solvent is DMF or DMA.
In a specific embodiment of the present invention, the fourth solvent is DCM, THF or CAN.
The synthesis method of SET0526 has high yield and conversion rate, is easy to amplify and is convenient for commercial production.
Drawings
FIG. 1 is an HPLC chromatogram of MA-PEG4-VC-PAB according to an embodiment of the invention.
FIG. 2 is a LCMS spectrum of MA-PEG4-VC-PAB according to an embodiment of the invention.
FIG. 3 is an HPLC chart of MA-PEG4-VC-PAB-PNP according to an embodiment of the present invention.
FIG. 4 is a LCMS spectrum of PMA-PEG4-VC-PAB-PNP according to an embodiment of the present invention.
FIG. 5 is a LCMS spectrum of PNU-Acid according to an embodiment of the present invention.
FIG. 6 is an HPLC chromatogram of PNU-EDA-Fmoc of an embodiment of the present invention.
FIG. 7 is a LCMS spectrum of PNU-EDA-Fmoc of an embodiment of the present invention.
FIG. 8 is an HPLC chromatogram of PNU-EDA of an embodiment of the present invention.
FIG. 9 is a LCMS spectrum of PNU-EDA of an embodiment of the present invention.
Fig. 10 is an HPLC profile of SET0526 of an embodiment of the present invention.
Fig. 11 is a LCMS spectrum of SET0526 of an embodiment of the present invention.
Fig. 12 is a HNMR spectrum of SET0526 according to an embodiment of the present invention.
Detailed Description
Example 1
Synthesis of Compound MA-PEG4-VC-PAB
MA-PEG4-C 2H4 COOH 222mg (1.2 eq) was dissolved under the protection of DMF 2mL nitrogen at room temperature with stirring, HATU 384mg (1.9 eq) was added, ice water was cooled to about 0 ℃, VC-PAB 200mg (1.0 eq) was added, DIPEA 170mg (2.5 eq) was added dropwise, and the mixture was allowed to naturally warm to room temperature under the protection of nitrogen and reacted for about 2-3 hours. LC-MS/HPLC detection, and the reaction of the raw materials is completed. Post-treatment: direct medium pressure reverse phase column purification (water/acetonitrile system purification, 30% acetonitrile ratio product), freeze drying to obtain compound MA-PEG4-VC-PAB 160mg, yield 43.0%, HPLC purity 97.1%, LCMS 707.74[ M+H ] +. The HPLC spectrum of MA-PEG4-VC-PAB is shown in FIG. 1, and the LCMS spectrum is shown in FIG. 2.
Synthesis of MA-PEG4-VC-PAB-PNP
160Mg (1.0 eq) of MA-PEG4-VC-PAB was dissolved in 2mL of DMF, 83mg (1.2 eq) of DNPC was added, and 59mg (2.0 eq) of DIPEA was reacted at room temperature with stirring for 16h. LC-MS/HPLC detection shows that the reaction of the raw materials is basically finished. Post-treatment: direct medium pressure reverse phase column purification (80 g reverse phase column, 220nm, water/acetonitrile system purification, 50% acetonitrile ratio product). The mixture was collected and lyophilized to give 144mg of MA-PEG4-VC-PAB-PNP in 73.0% yield, HPLC (FIG. 3): 99.4%, LCMS (FIG. 4): 872.86[ M+H ] +.
Synthesis of PNU-Acid
500Mg (1.0 eq) of Compound 6 was taken in a 50mL single-necked flask, 5mL of THF was added, the temperature was lowered to-5℃and stirred, 0.86mL (1.3 eq) of TBAF (1 Min THF) was taken and added dropwise into the reaction flask, the reaction was completed for about 20Min, 2.5mL of water and 5mL of trifluoroethanol were added, 149mg (1.05 eq) of sodium periodate was added, the reaction was carried out to 0℃for about 2h, and the reaction was completed by LCMS. Post-treatment: 5mL of water was added, THF was removed by rotary evaporation, and the crude product was lyophilized to give a crude product which was used directly in the next reaction, LCMS as shown in FIG. 5: 628.43[ M+H ] +.
Synthesis of PNU-EDA-Fmoc
5MDMF was added to dissolve the PNU-Acid crude product (theory: 418 mg,1.0 eq), 89.8mg (1.0 eq) of HOAT, prBrOP mg (2.0 eq) of ice bath were added, the temperature was lowered to 0-5 ℃ and stirred for 5min, 316mg (2.0 eq) of EDA-Fmoc and 427mg (5.0 eq) of DIPEA were added, the temperature was naturally raised to 20-25 ℃ for reaction for about 2h, and LC-MS/HPLC detection was performed, after the reaction of the raw materials was completed. Post-treatment: medium pressure reverse phase column purification (80 g reverse phase column, 234nm, water/acetonitrile system purification, 700% acetonitrile ratio product). The resulting mixture was collected and lyophilized to give 200mg of PNU-EDA-Fmoc in 33.9% two-step yield, HPLC (FIG. 6): 94%, LCMS (FIG. 7): 892.69[ M+H ] +.
Synthesis of the Compound PNU-EDA
214Mg (1.0 eq) PNU-EDA-Fmoc was dissolved by adding 4mL DCM/1mL ACN, cooled to 0-5℃in ice bath and stirred for 5min, 0.48mL DBU (1M in ACN) was added (2.0 eq), naturally warmed to room temperature and stirred for 1h at 20-25 ℃. LC-MS/HPLC detection, and the reaction of the raw materials is completed. Post-treatment: 1mL of 0.05% ammonium bicarbonate water was added, the organic phase was removed by flash evaporation, followed by medium pressure reverse phase column purification ((80 g reverse phase column, 234nm,0.05% ammonium bicarbonate water/acetonitrile system, 30% acetonitrile ratio product)), and lyophilization was collected to give PNU-EDA80mg in 49.8% yield, HPLC as shown in FIG. 8 at 94% and LCMS as shown in FIG. 9 at 670.57[ M+H ] +.
Synthesis of SET0526 (MA-PEG 4-VC-PAB-EDA-PNU 159582)
39Mg (1.0 eq) of MA-PEG4-VC-PAB-PNP, 33mg (1.2 eq) of PNU-EDA is dissolved in 1mL of DMF, cooled to 0-5 ℃ in an ice bath, stirred for 5min, 6.1mg (1.9 eq) of HOBt and 12mg (2.5 eq) of DIPEA are added, the temperature is naturally raised to 20-25 ℃ for reaction for about 2h, the detection is carried out by LC-MS/HPLC, and the reaction of the raw materials is completed. Post-treatment: high pressure liquid phase preparation, 234nm, flow rate 50mL/min,0.05% NH4HCO3 water/acetonitrile, 60% acetonitrile ratio out product, collecting product, freeze drying to obtain SET0526 30mg, yield 47.8%, HPLC (see FIG. 10): 92.49%, LCMS (FIG. 11): 1403.16[ M+H ] +.
As shown in fig. 12 1H NMR(400MHz,DMSO)δ14.06(s,1H),13.24(s,1H),9.97(s,1H),8.10(d,J＝6.9Hz,3H),7.89(dd,J＝20.3,6.6Hz,4H),7.71-7.57(m,4H),7.32-7.22(m,4H),7.02(s,2H),5.98(t,J＝5.6Hz,1H),5.40(s,2H),5.27(dd,J＝11.7,7.3Hz,2H),5.04-4.94(m,4H),4.59(d,J＝2.0Hz,1H),4.38(d,J＝5.4Hz,1H),4.26-4.19(m,3H),4.13(d,J＝7.2Hz,1H),4.00(s,3H),3.93(d,J＝7.6Hz,1H),3.66(d,J＝10.3Hz,1H),3.62-3.58(m,2H),3.52(d,J＝5.0Hz,3H),3.47(d,J＝5.8Hz,12H),3.35(s,110H),3.24-3.18(m,3H),3.14-3.09(m,3H),3.04-2.88(m,6H),2.73-2.59(m,3H),2.39(dd,J＝13.6,7.1Hz,2H),2.16(d,J＝12.9Hz,1H),2.03-1.92(m,2H),1.78-1.55(m,6H),1.46-1.34(m,3H),1.26-1.18(m,6H),1.13(d,J＝6.4Hz,1H),0.85(dd,J＝12.8,6.8Hz,8H).
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (2)
1. A method for synthesizing an antibody-conjugated drug intermediate SET0526, comprising the steps of:
Stirring the compound MA-PEG4-C 2H4 COOH and HATU in a first solvent, cooling to 0-5 ℃ with ice water, adding the compound VC-PAB and DIPEA, reacting for 2-3 h under the protection of nitrogen, passing the reaction solution through a reverse phase column, and purifying to obtain the compound MA-PEG4-VC-PAB; the combined molar ratio of MA-PEG4-C 2H4 COOH, HATU, first solvent, VC-PAB and DIPEA was 1.0eq:1.0eq-1.5eq:10V-20V:1.0eq:2.0eq-2.5eq;
Dissolving the compound MA-PEG4-VC-PAB in a first solvent, adding DNPC and DIPEA, reacting for 14-16 hours at room temperature, purifying by medium-pressure reverse phase column chromatography, and obtaining the compound MA-PEG4-VC-PAB-PNP; the mixed molar ratio of MA-PEG4-VC-PAB, DNPC and DIPEA in the first solvent was 1.0eq:10V-20V:1.0eq-1.5eq:2.0eq-2.5eq;
Dissolving a compound PNU-TBS in a second solvent, cooling to-5-0 ℃, adding TBAF, reacting for 20-40min, adding water and trifluoroethanol, adding sodium periodate, heating to 0-5 ℃ for reacting for 2-4h, detecting that the raw materials are completely reacted, directly concentrating and freeze-drying the reaction solution, and obtaining a crude compound PNU-Acid; the mixed molar ratio of PNU-TBS, in the second solvent, TBAF, water, trifluoroethanol, sodium periodate was 1.0eq:10V-20V:1.0eq-3.0eq:10V-20V:1.0eq-1.1eq;
Dissolving the crude compound PNU-Acid in a third solvent, sequentially adding HOAt and PrBrOP, cooling to 0-5 ℃ in an ice bath, stirring for 5-20min, adding EDA-Fmoc and DIPEA, naturally heating to 20-25 ℃ for reacting for 2-4h, carrying out aftertreatment, carrying out medium-pressure reverse phase column purification, and freeze-drying to obtain the compound PNU-EDA-Fmoc; the mixed molar ratio of PNU-Acid, third solvent, HOAt, prBrOP, EDA-Fmoc and DIPEA was 1.0eq:10V-20V:1.0eq-2.0eq:1.0eq-2.5eq:1.0eq-2.5eq:3.0eq-6.0eq;
adding a compound PNU-EDA-Fmoc into a fourth solvent for dissolution, carrying out ice bath to 0-5 ℃ and stirring for 5-20min, adding DBU, naturally heating to room temperature of 20-25 ℃ and stirring for 1-2h, carrying out aftertreatment, concentrating a reaction solution, carrying out medium-pressure reversed-phase column purification and freeze-drying to obtain the compound PNU-EDA; the mixed molar ratio of PNU-EDA-Fmoc, fourth solvent, DBU was 1.0eq:10V-30V:1.0eq-3.0eq;
Dissolving a compound MA-PEG4-VC-PAB-PNP and PNU-EDA in a third solvent, adding HOBt and DIPEA, naturally heating to 20-25 ℃ for reacting for 2-4h, and performing post-treatment to obtain SET0526; the mixing molar ratio of MA-PEG4-VC-PAB-PNP, PNU-EDA, third solvent, HOBt, DIPEA was 1.0eq:1.0eq-2.0eq:10V-30V:1.0eq-2.0eq:2.0eq-4.0eq;
The first solvent is DCM, THF or DMF, the second solvent is DCM or THF, the third solvent is DMF or DMA, and the fourth solvent is DCM, THF or CAN.
2. The synthetic method of claim 1, wherein the post-treatment when preparing SET0526 comprises the steps of:
High pressure liquid phase preparation, 234nm, flow rate 50mL/min,0.05% NH 4HCO3 water/acetonitrile, lyophilization.
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